Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol.

2-(Dimethylamino)ethan-1-ol (Deanol) is a widely produced chemical used by both industry and consumers in a variety of applications. Meclofenoxate, a stimulant classified on the World Anti-Doping Agency Prohibited List, metabolizes into deanol and, presumably, its main metabolite deanol-N-oxide. Hence, using liquid chromatography-tandem mass spectrometry, a quantitative detection method for deanol-N-oxide in urine was developed. Subsequently, the urinary excretion of deanol-N-oxide after oral application of 130 mg of deanol was determined in six volunteers, and urine samples of a cohort of 180 male and female athletes from different sports were analyzed. In addition, urinary deanol-N-oxide was determined in an exploratory study with one volunteer ingesting 250 mg of meclofenoxate. The developed test method allowed for limits of detection and quantification for deanol-N-oxide at 0.05 and 0.15 µg/mL, respectively. Urinary deanol-N-oxide cmax levels were found between 100 and 250 µg/mL 2-5 h post-administration of 130 mg of deanol. Similarly, urine samples collected after the administration of 250 mg of meclofenoxate exhibited cmax levels of 115 µg/mL. In contrast, deanol-N-oxide urine concentrations of pre-administration specimens and 180 routine doping control urine sample were between 0.3 and 1.3 µg/mL and below limit of quantification and 1.8 µg/mL, respectively. The study suggests that the use of deanol and meclofenoxate results in significantly elevated urinary deanol-N-oxide levels. Whether or not monitoring deanol-N-oxide in doping controls can support decision-making processes concerning the detection of meclofenoxate use necessitates further investigations taking into consideration the elimination kinetics of 4-chlorophenoxyacetic acid, the main metabolite of meclofenoxate, and deanol-N-oxide.

Usefulness of serum androgen isotope ratio mass spectrometry (IRMS) to detect testosterone supplementation in women.

The detection of testosterone intake is facilitated by monitoring the urinary steroid profile in the athlete biological passport. This technique can be used with confidence to identify target samples for isotope ratio mass spectrometry. Regrettably, most research has been performed on male subjects resulting in a method that does not account for females' steroid concentration and/or variation. This study evaluates the usefulness of the carbon isotope ratio (CIR) in serum of female subjects. Two steroid sulphates are targeted in serum, androsterone and epiandrosterone. Both exhibit statistically significant depletion of their CIR after 10 weeks of daily (10 mg) transdermal testosterone administration. Of the 21 female subjects, samples from six individuals were identified as adverse analytical findings; additionally, four were found atypical considering the serum CIR. The urinary athlete biological passport was not sufficiently sensitive to identify target serum samples for isotope ratio mass spectroscopy. Of the six with a suspicious passport, only two could be confirmed using the serum CIR of androsterone and epiandrosterone. This study shows that CIR analysis in serum cannot be considered the sole confirmatory solution to detect testosterone doping in women due to low sensitivity. However, this analysis has the potential to be used as a complementary method in certain situations to confirm exogenous testosterone in women.

Addressing recent challenges in isotope ratio mass spectrometry: Development of a method applicable to 1-androstene-steroids, 6α-hydroxy-androstenedione, and androstatrienedione.

In 2020, the confirmation of the non-endogenous origin of several pseudo-endogenous steroids by means of isotope ratio mass spectrometry (IRMS) was recommended by the World Anti-Doping Agency (WADA), in addition to previously established target analytes for IRMS in sports drug testing. To date, however, IRMS-based methods validated in accordance with current WADA regulations have not been available. Therefore, the aim of this research project was the development and validation of a method to determine the carbon isotope ratios (CIR) of all newly considered pseudo-endogenous steroids, encompassing the anabolic androgenic steroids comprising a 1-ene-core structure (5α-androst-1-ene-3ß,17ß-diol, 5α-androst-1-ene-3,17-dione [1AD], 17ß-hydroxy-5α-androst-1-en-3-one, 3α-hydroxy-5α-androst-1-ene-17-one [1AND], and 3ß-hydroxy-5α-androst-1-ene-17-one [1EpiAND]), as well as steroids referred to as hormone and metabolic modulators (androsta-1,4,6-triene-3,17-dione [TRD] and its main metabolite 17ß-hydroxy-androsta-1,4,6-triene-3-one) and 6α- and 6ß-hydroxy-androst-4-ene-3,17-dione. With peak purity of target analytes being critical for IRMS analyses, a twofold high-performance liquid chromatography (HPLC)-based sample purification was employed, with all analytes being acetylated between the first and second HPLC fractionation. Using established gas chromatography/combustion/IRMS instrumentation, limits of quantification were estimated at 10 ng/ml for a 20 ml urine aliquot for all analytes, except for 1AND (20 ng/ml), and combined measurement uncertainties were estimated between 0.4‰ and 0.9‰. For proof-of-concept, samples collected after the single oral administration of a nutritional supplement containing 1AD and 1EpiAND were analyzed as well as existing excretion study urine samples obtained after the administration of 4-androstenedione and TRD. Based on the obtained results, the developed method was considered to be fit-for-purpose.

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis.

RATIONALE: The misuse of 7-oxo-DHEA (3ß-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7ß-OH-DHEA were identified, necessitating further investigations. METHODS: As both 7-oxo-DHEA and 7ß-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA. RESULTS: Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7ß-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5α-androstane-3ß,7ß-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7ß-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7ß-OH-DHEA. CONCLUSIONS: As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7ß-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens.

Quantifying cobalt in doping control urine samples--a pilot study.

Since first reports on the impact of metals such as manganese and cobalt on erythropoiesis were published in the late 1920s, cobaltous chloride became a viable though not widespread means for the treatment of anaemic conditions. Today, its use is de facto eliminated from clinical practice; however, its (mis)use in human as well as animal sport as an erythropoiesis-stimulating agent has been discussed frequently. In order to assess possible analytical options and to provide relevant information on the prevalence of cobalt use/misuse among athletes, urinary cobalt concentrations were determined by inductively coupled plasma-mass spectrometry (ICP-MS) from four groups of subjects. The cohorts consisted of (1) a reference population with specimens of 100 non-elite athletes (not being part of the doping control system), (2) a total of 96 doping control samples from endurance sport athletes, (3) elimination study urine samples collected from six individuals having ingested cobaltous chloride (500 µg/day) through dietary supplements, and (4) samples from people supplementing vitamin B12 (cobalamin) at 500 µg/day, accounting for approximately 22 µg of cobalt. The obtained results demonstrated that urinary cobalt concentrations of the reference population as well as the group of elite athletes were within normal ranges (0.1-2.2 ng/mL). A modest but significant difference between these two groups was observed (Wilcoxon rank sum test, p < 0.01) with the athletes' samples presenting slightly higher urinary cobalt levels. The elimination study urine specimens yielded cobalt concentrations between 40 and 318 ng/mL during the first 6 h post-administration, and levels remained elevated (>22 ng/mL) up to 33 h. Oral supplementation of 500 µg of cobalamin did not result in urinary cobalt concentrations > 2 ng/mL. Based on these pilot study data it is concluded that measuring the urinary concentration of cobalt can provide information indicating the use of cobaltous chloride by athletes. Additional studies are however required to elucidate further factors potentially influencing urinary cobalt levels.

Metabolism of androsta-1,4,6-triene-3,17-dione and detection by gas chromatography/mass spectrometry in doping control.

The urinary metabolism of the irreversible aromatase inhibitor androsta-1,4,6-triene-3,17-dione was investigated. It is mainly excreted unchanged and as its 17beta-hydroxy analogue. For confirmation, 17beta-hydroxyandrosta-1,4,6-trien-3-one was synthesized and characterized by nuclear magnetic resonance (NMR) in addition to the parent compound. In addition, several reduced metabolites were detected in the post-administration urines, namely 17beta-hydroxyandrosta-1,4-dien-3-one (boldenone), 17beta-hydroxy-5beta-androst-1-en-3-one (boldenone metabolite), 17beta-hydroxyandrosta-4,6-dien-3-one, and androsta-4,6-diene-3,17-dione. The identification was performed by comparison of the metabolites with reference material utilizing gas chromatography/mass spectrometry (GC/MS) of the underivatized compounds and GC/MS and GC/tandem mass spectrometry (MS/MS) of their trimethylsilyl (TMS) derivatives. Alterations in the steroid profile were also observed, most obviously in the androsterone/testosterone ratio. Even if not explicitly listed, androsta-1,4,6-triene-3,17-dione is classified as a prohibited substance in sports by the World Anti-Doping Agency (WADA) due to its aromatase-inhibiting properties. In 2006 three samples from human routine sports doping control tested positive for metabolites of androsta-1,4,6-triene-3,17-dione. The samples were initially found suspicious for the boldenone metabolite 17beta-hydroxy-5beta-androst-1-en-3-one. Since metabolites of androst-4-ene-3,6,17-trione were also present in the urine samples, it is presumed that these findings were due to the administration of a product like 'Novedex Xtreme', which could be easily obtained from the sport supplement market.

Determination of the origin of urinary norandrosterone traces by gas chromatography combustion isotope ratio mass spectrometry.

On the one hand, 19-norandrosterone (NA) is the most abundant metabolite of the synthetic anabolic steroid 19-nortestosterone and related prohormones. On the other hand, small amounts are biosynthesized by pregnant women and further evidence exists for physiological origin of this compound. The World Anti-Doping Agency (WADA) formerly introduced threshold concentrations of 2 or 5 ng of NA per ml of urine to discriminate 19-nortestosterone abuse from biosynthetic origin. Recent findings showed however, that formation of NA resulting in concentrations in the range of the threshold levels might be due to demethylation of androsterone in urine, and the WADA 2006 Prohibited List has defined NA as endogenous steroid. To elucidate the endogenous or exogenous origin of NA, (13)C/(12)C-analysis is the method of choice since synthetic 19-nortestosterone is derived from C(3)-plants by partial synthesis and shows delta(13)C(VPDB)-values of around -28 per thousand. Endogenous steroids are less depleted in (13)C due to a dietary mixture of C(3)- and C(4)-plants. An extensive cleanup based on two high performance liquid chromatography cleanup steps was applied to quality control and doping control samples, which contained NA in concentrations down to 2 ng per ml of urine. (13)C/(12)C-ratios of NA, androsterone and etiocholanolone were measured by gas chromatography/combustion/isotope ratio mass spectrometry. By comparing delta(13)C(VPDB)-values of androsterone as endogenous reference compound with NA, the origin of NA in doping control samples was determined as either endogenous or exogenous.